Tetanus toxin is a protein neurotoxin of 1315 amino acids. The mechanisms by which tetanus toxin binds to and enters cells, and inhibits neurosecretion are not well understood. Fragment C is an approximately 50,000 M.W. peptide derived by proteolysis from the carboxy terminus of tetanus toxin that retains the ability to bind to gangliosides present in neuronal cell membranes. We are defining specific domains and amino acids of Fragment C which are required for recognizing and binding to cellular receptors. Peptides corresponding to specific regions of Fragment C were expressed from polymerase chain reaction amplified clostridial DNA using in vitro transcription and translation systems, and studied for ganglioside binding activity. Full length Fragment C (458 amino acids) produced by this method resembled tetanus toxin-derived Fragment C in its ganglioside binding properties. Deletions of up to 263 amino acids from the amino terminal of Fragment C did not affect binding. Deletion of five amino acids from the carboxy terminus did not affect binding, however, deletion of ten or more amino acids resulted in complete loss of binding activity. In other studies, an antibody produced against the carboxy terminal 20 amino acids of tetanus toxin was demonstrated to inhibit the binding of 125I-Fragment C to gangliosides. These data suggest that the carboxy terminal region of tetanus toxin is required for receptor recognition, either as a component of the ganglioside recognition site or as a domain necessary for retention of a functional conformation.